Thermocycling system, composition, and microfabrication method

ABSTRACT

A system and method of manufacture for the system, comprising a set of heater-sensor dies, each heater-sensor die comprising an assembly including a first insulating layer, a heating region comprising an adhesion material layer coupled to the first insulating layer and a noble material layer, and a second insulating layer coupled to the heating region and to the first insulating layer through a pattern of voids in the heating region, wherein the pattern of voids in heating region defines a coarse pattern associated with a heating element of the heating region and a fine pattern, integrated into the coarse pattern and associated with a sensing element of the heating region; an electronics substrate configured to couple heating elements and sensing elements of the set of heater-sensor dies to a controller; and a set of elastic elements configured to bias each of the set of heater-sensor dies against a detection chamber.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/879,513 filed 18 Sep. 2013, which is incorporated in its entiretyherein by this reference.

TECHNICAL FIELD

This invention relates generally to the molecular diagnostics field, andmore specifically to an improved sample thermocycling system andfabrication process thereof.

BACKGROUND

Molecular diagnostics is a clinical laboratory discipline that hasdeveloped rapidly during the last 25 years. It originated from basicbiochemistry and molecular biology research procedures, but now hasbecome an independent discipline focused on routine analysis of nucleicacids (NA), including deoxyribonucleic acid (DNA) and ribonucleic acid(RNA) for diagnostic use in healthcare and other fields involvinganalysis of nucleic acids. Molecular diagnostic analysis of biologicalsamples can include the detection of one or more nucleic acid materialspresent in the specimen. The particular analysis performed may bequalitative and/or quantitative. Methods of analysis typically involveisolation, purification, and amplification of nucleic acid materials,and polymerase chain reaction (PCR) is a common technique used toamplify nucleic acids. Often, a nucleic acid sample to be analyzed isobtained in insufficient quantity, quality, and/or purity, hindering arobust implementation of a diagnostic technique. Current sampleprocessing methods and molecular diagnostic techniques are oftenlabor/time intensive, low throughput, and expensive, and systems ofanalysis are insufficient.

A rapid and efficient thermocycling system that can reliably thermocyclereagents used for processing of nucleic acids can significantly improvethe efficiency and effective implementation of molecular diagnostictechniques, such as realtime polymerase chain reaction (RT-PCR).Microfabrication techniques can produce such thermocycling systemscomprising precision heaters with low thermal masses and withwell-coupled temperature sensors. However, challenges are inherent inensuring that the microfabrication and assembly processes utilized tofabricate thermal cycling elements are extremely robust and reliable.

Due to these challenges and deficiencies of current molecular diagnosticsystems and methods, there is thus a need for an improved samplethermocycling system and fabrication process thereof. This inventionprovides such a system and fabrication process.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B depict embodiments of a thermocycling system;

FIG. 2 depicts an example schematic of a heater-sensor die duringfabrication in an example of a thermocycling system;

FIG. 3 depicts an example of heating and sensing elements in an exampleof a thermocycling system;

FIGS. 4A and 4B depict examples of additional elements of an embodimentof a thermocycling system;

FIGS. 5A and 5B depict variations of configurations of elements in anembodiment of a thermocycling system;

FIGS. 6A and 6B depict variations of configurations of elements in anembodiment of a thermocycling system;

FIGS. 7A-7C depict variations in configurations of additional elementsin an embodiment of a thermocycling system; and

FIG. 8 depicts a schematic of an embodiment of a method formanufacturing a thermocycling system.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following description of the preferred embodiments of the inventionis not intended to limit the invention to these preferred embodiments,but rather to enable any person skilled in the art to make and use thisinvention.

1. System

As shown in FIGS. 1A and 1B, an embodiment of a system 100 forthermocycling biological samples within detection chambers comprises: aset of heater-sensor dies 110; an electronics substrate 140 that couplethe set of heater-sensor dies to a controller; a set of heat sinksupports 150 coupled to at least one of the electronics substrate andthe set of heater-sensor dies; and a set of elastic elements 160 coupledto the electronics substrate and configured to bias each of the set ofheater-sensor dies against a detection chamber. In some embodiments, thesystem 100 further comprises a controller 165 and/or a cooling subsystem170 configured to actively cool the system 100.

The system 100 functions to enable rapid thermal cycling of sampleswhile providing uniform heating and preventing signal drift. In specificapplications, the system 100 can be used to rapidly and controllablythermocycle nucleic acid samples during performance of moleculardiagnostic amplification techniques (e.g., PCR, RT-PCR), signalamplification techniques (e.g., bDNA, hybrid capture), and analyticaltechniques (e.g., gel electrophoresis, mass spectrometry). The system100 can also provide rapid thermocycling without significant powerrequirements, ensure a closer correlation between the actual heatingtemperature and the temperature set-point by implementing an integratedheater-sensor die, and controllably and individually heat small samplevolumes (e.g., picoliters, nanoliters) based upon a microfabricationtechnique that also enables mass production of the system 100.Furthermore, the system 100 implements a priori predictions ofelectrical resistance values of thin film combinations of the set ofheater-sensor dies 110, and accounts for and/or prevents signal drift tomaintain controlled sample heating. In some variations, the system 100can be integrated into a molecular diagnostic system, such as thatdescribed in U.S. Pub. No. 2013/0210015, entitled “System and Method forProcessing and Detecting Nucleic Acids”, and filed on 13 Feb. 2013;however, the system 100 can additionally or alternatively be used withany other suitable system for processing biological or non-biologicalsamples.

1.1 Heater-Sensor Dies

The set of heater-sensor dies 110 functions to controllably heatindividual sample volumes. Preferably, each heater-sensor die 111 is athin-film die that can be deposited onto another substrate (e.g.,silicon, glass substrate) that can be packaged onto an electronicssubstrate 140 (e.g., printed circuit board, PCB); however, eachheater-sensor die 111 can alternatively comprise any suitable geometryand/or configuration that enables controlled, uniform, and rapid heatingof a detection chamber in thermal communication with the heater-sensordie 111. In some embodiments, the detection chambers can be thosedescribed in U.S. Pub. No. 2013/0209326, entitled “MicrofluidicCartridge for Processing and Detecting Nucleic Acids” and filed on 13Feb. 2013, which is herein incorporated in its entirety by thisreference; however, the detection chambers can alternatively be anyother suitable container for processing a biological sample. Preferably,each heater-sensor die 111 is characterized by a small profile (e.g.,<100 mm dimension), which ensures that the heater-sensor die 111 is ableto thermocycle rapidly; however, a heater-sensor die 111 canalternatively be characterized by any suitable profile in order to meetany other thermocycling requirement. Additionally, each heater-sensordie 111 in the set of heater-sensor dies 110 is preferably configured toconform to a detection chamber (e.g., sample tube, sample container,sample heating zone of a cartridge for processing samples) configured tocontain a sample during heating; however, a heater-sensor die 111 in theset of heater-sensor dies 110 can alternatively not conform to adetection chamber for processing of a biological sample.

Preferably, each heater-sensor die 111 in the set of heater sensor dies110 comprises an assembly including: a first insulating layer 112 a thatfunctions to provide an insulating barrier to isolate the heaters andsensors and a heating region 113 that functions to provide uniformsample heating. Some variations can further include a second insulatinglayer 112 b that functions to provide an additional insulating barrierthat opposes the first insulating layer 112 a. The insulating layer(s)112 a, 112 b are preferably electrically insulating, but canadditionally be thermally insulating in variations where localizedheating is also desired. Furthermore, each heater-sensor die inpreferably comprises two insulating layers 112 a, 112 b that areconfigured to “sandwich” the heating region 113, thus isolating theheating region 113 at least at two surfaces of the heating region 113;however, each heater-sensor dies 111 can alternatively comprise anysuitable number of insulating layers 112 arranged relative to theheating region 113 in any suitable manner. In one variation, asdescribed in further detail below, the heating region 113 can include apattern of voids that defines elements of the heating region, and asecond insulating layer 112 b can be configured to couple to the heatingregion 113 and to the first insulating layer 112 a through the patternof voids in the heating region 113, as shown in FIG. 2. The secondinsulating layer 112 b can, however, be configured to couple to theheating region 113 and/or to the first insulating layer 112 a in anyother suitable manner.

The heating region 113 preferably comprises a heating element 114 withan integrated sensing element 115, as shown in FIG. 3, and is composedof at least one metal or metal alloy, with configurations described infurther detail below. Furthermore, the heating region 113 preferablydefines a pattern 313 of voids having geometric features (e.g., width,thickness, length, spacing) that facilitate uniform heating and providedesired heating and sensing characteristics (e.g., resistancecharacteristics). In some variations, the pattern 313 can comprise anyone or more of: linear segments, non-linear segments, boustrophedonicsegments, continuous segments, non-continuous segments, and any othersuitable segment(s) having any other suitable geometry (i.e., width,depth, height, length, path, etc.). The pattern 313 can be symmetricabout any suitable reference (e.g., reference line, reference plane,etc.), or can alternatively be non-symmetric. Furthermore, in somevariations, the pattern 313 can define a global morphology (e.g.,circular footprint, ellipsoidal footprint, polygonal footprint, etc.) ata first scale (e.g., macroscopic scale) but have a local morphology at asecond scale (e.g., microscopic scale), wherein the local morphologyprovides desired characteristics (e.g., resistance characteristics) thatare attributed to elements (e.g., heating elements 114, sensing elements115) of the heating region 113. As such, the global morphology canprovide conformation (e.g., in shape) between the heating region 113 anda detection chamber coupled to or corresponding to a heater-sensor dies111, and the local morphology can provide uniform heating and/oraccurate sensing of heating parameters by utilizing structural featureshaving a smaller governing dimension.

In a specific example, as shown in FIG. 3, the heating elements 114 a,114 b, 114 c with integrated sensing elements 115 a, 115 b, 115 c aredefined by a pattern 313 with a global morphology characterized by acircular footprint, but a local morphology of structures characterizedby a continuous boustrophedonic arrangement, wherein both the globalmorphology and local morphology were designed according to theory andfabricated as described in further detail below. In the specificexample, the sensing element 115 is configured to be centrally locatedwithin the global morphology of the heating element 114. However, inalternative variations, the heating region 113 can alternatively notcomprise an integrated sensing element 115, can comprise any suitablenumber of heating elements 114/sensing elements 115, and/or can becomposed of any other suitable material.

Furthermore, during processing, each heater-sensor die 111 in the set ofheater-sensor dies 110 is preferably annealed, as described in furtherdetail below, to improve adhesion and settling of the heater-sensormaterials (e.g., on a silicon substrate). The annealing can comprise asingle stage of annealing or multiple stages of annealing, and canadditionally or alternatively comprise different conditions (e.g.,temperatures, durations, environmental conditions) during the annealingstage(s). To exemplify an effect that changes in resistance behavior canhave upon heating and sensing function, a ˜1-2Ω deviation in resistancecan cause significant deviations (e.g., 5-10° C.) between intended andactual temperatures attained by a heater-sensor dies 111. Suchdeviations in temperature can lead to unwanted variability in moleculardiagnostic technique results and/or unreliable data (e.g., falsepositives, false negatives, indeterminate results). Even further,temperature swings greater than 10° C. can result in permanent damage tothe thermocycling system 100 and/or to a sample being processed usingthe system 100. Given the importance of maintaining the calibration ofthe sensing elements 115 over long periods of time, it is preferable toensure that minimal dynamics occur in the materials of the heater-sensordies 111 after fabrication is complete. Since a majority of changes inresistance (e.g., drift) come from thermal reactions, settling,diffusion, and improved adhesion, annealing can accelerate the processesthat lead to changes in resistance, thus producing heater-sensor dies111 with stable resistance behavior (e.g., stable for 3-5 years) bydriving dynamic processes toward an equilibrium state (or other stablestate) prior to use of the heater-sensor dies 111 in sample-processingapplications. Preferably, annealing thus produces stable resistancesthat are substantially low (in order to enable driving at low voltages)and have limited variability, indicating that underlying materialdynamics have reached equilibrium. Alternatively, during processing,each heater-sensor die 111 in the set of heater-sensor dies 110 may notbe annealed, can comprise elements that limit temperature swings, and/ormay undergo any other suitable process to produce stable resistancebehavior in any other suitable manner.

In one variation, as shown in FIG. 2, the insulating layers 112 a, 112 bof the heater-sensor dies 111 are composed of an oxide (e.g., siliconoxide), and the heating region 113 comprises an adhesion material layer116 coupled to the first insulating layer 112 a and configured topromote adhesion of additional deposited layers (e.g., a noble materiallayer 117) of the heating region 113, and at least one noble materiallayer 117 coupled to the adhesion material layer 116 and configured toreduce or prevent signal drift that can result due to diffusion of theadhesion layer 116. In this variation, the first insulating layer 112 ais grown or deposited uniformly on the surface of a silicon wafer (e.g.,by thermal oxidation), the material layers 116, 117 of the heatingregion 113 are deposited upon the first insulating layer 112 a (e.g., byevaporation, by sputtering, etc.), the heating region 113 is etched todefine the heater/sensor pattern 313 of the heating element 114 and thesensing element 115 (e.g., by lithography using a positive resist, bylithography using a negative resist), and a second insulating layer 112b is deposited on the heating region 113 (e.g., by chemical vapordeposition) to insulate the heating region 113 between two insulatingoxide layers. Preferably, the adhesion materials used in the adhesionmaterial layer(s) 116 comprise materials that are oxygen-active to react(e.g., chemically react) with an oxide surface (e.g., of an insulatinglayer 112 a, 112 b). In examples, the adhesion material layer 116 can becomposed of any one or more of: chromium, titanium, niobium, vanadiumand any combination or alloy thereof. In examples, the noble materiallayer can be composed of any one or more of: gold, platinum, tungsten,palladium, and any combination or alloy thereof. Furthermore, variationsof the examples can include any suitable number of adhesion materiallayer(s) 116 and/or noble material layer(s) 117 coupled between theinsulating layer(s) 112 a, 112 b, wherein each layer 116, 117 can haveany other suitable thickness.

In a first specific example of this variation, as shown in FIG. 3, aheater-sensor die 111 is configured to uniformly heat a circular regionhaving a diameter of 5 mm, the heater-sensor dies 111 has a footprintspanning a region of ˜8.6 mm×7 mm, and the heating region 113 of theheater-sensor dies 111 defines three heating elements 114 a, 114 b, 114c: a central circular heating element 114 a and two circumferentialheating elements 114 b, 114 c configured to form a boundary with aserpentine-shaped pattern about the central circular heating element 114a. In the first specific example, the two circumferential heatingelements 114 b, 114 c each form a semicircular perimeter about thecentral circular heating element 114 a. The heater-sensor die 111 in thefirst specific example further comprises three integrated sensingelements (i.e., resistance temperature sensors, RTDs) distributed atthree locations within the 5 mm circular region. In the first specificexample, the heating elements 114 comprise a 50 nm chromium adhesionmaterial layer 116 and a 200 nm gold noble material layer 117 and wereetched away in a boustrophedonic pattern to define the pattern of voids313, designed using a layout editor (e.g., Mentor Graphics™ or L-Edit™),to form the heating elements 114 and the sensing elements 115. Theheating elements 114 a, 114 b, 114 c are defined by coarse patterning inthe first specific example, and the sensing elements 115 are integratedwith the heating elements 114 a, 114 b, and 114 c and defined by finepatterning, as shown in FIGS. 3A and 3B. In the first specific example,the heating elements 114 are characterized with resistances in the rangeof 40-100Ω and the accompanying sensing elements 115 (i.e., RTDs) arecharacterized with resistances in the range of 200-250Ω prior toannealing. With annealing at 400 F in an inert N₂ atmosphere, theresistances for both the heating elements 114 and the sensing elements115 increased 400-600% after 1 hour, and increased up to 800% withincreasing anneal times.

In other embodiments of the heater-sensor dies 111, the heatingelement(s) 114 and/or the sensing element(s) 115 can comprise othercombinations of adhesion material layer(s) 116 and/or noble materiallayer(s) 117 in addition to or alternative to chromium and gold,including any one or more of: titanium (adhesion), platinum (noble),tungsten (noble), and any combination or alloy thereof. In particular,chromium and titanium can serve as preferable adhesion materials for anactive noble layer being coupled to the adhesion material layer 116, dueto attributes that enable them to react with oxide materials. Thecombination of chromium and gold additionally can undergo processing bya lift-off method or an etching process to form a heating element 114and/or a sensing element 115, as described in further detail in Section2 below. While in some applications it can be difficult to etch platinumfilms due to platinum's non-reactivity, platinum has a preferabletemperature coefficient of resistance (TCR) of ˜0.00385 Ω/Ω/C that makesit stable and preferable for RTD fabrication. Platinum and/or titaniumcan even serve as an intermediate buffer layer 119, configured betweenan adhesion material layer 116 and a noble material layer 117, that canprevent diffusion from an adhesion material layer 116 into a noblematerial layer 117 and stabilize resistance behavior (e.g., electricalresistance dynamics). As such, variations of the heating region 113 caninclude any suitable number of intermediate buffer layers 119 thatprevent diffusion between an adhesion material layer 116 into anadjacent noble material layer 117.

In a second specific example, the heating elements 114 comprise a 50 nmchromium adhesion material layer 116, an 100 nm platinum intermediatebuffer layer 119 coupled to the adhesion material layer 116, and a 300nm gold noble material layer 117 coupled to the intermediate bufferlayer 119 and were etched away in an identical pattern to that in thefirst specific example to form a surface including the heating andsensing elements 114, 115. Similar to the first specific example, theheating elements 114 of the second specific example are defined bycoarse patterning, and the sensing elements 115 are defined by finepatterning and integrated into the coarse patterning of the heatingelements 114. Also similar to the first specific example, the heatingregion 113 is configured between two insulating layers 112 a, 112 b inthe second specific example. In the second specific example, the heatingelements 114 were characterized to have resistances of approximately 50Ωand the sensing elements were characterized to have resistances around130Ω prior to annealing. After annealing at 400 F in an inert N₂atmosphere, the resistances for both the heating elements 114 and thesensing elements increased 10-30% after 1 hour, with no substantialfurther change in resistance following additional annealing of 2 hours.

In a third specific example, the heating elements 114 comprise a 50 nmchromium adhesion material layer 116 and a 100 nm platinum noblematerial layer 117 coupled to the adhesion material layer 116 and wereetched away in an identical pattern to that in the first specificexample, to form a surface including the heating and sensing elements114, 115. Similar to the first specific example, the heating elements114 of the third specific example are defined by coarse patterning, andthe sensing elements 115 are integrated with the coarse patterning ofthe heating elements 114 and defined by fine patterning. Also similar tothe first specific example, the heating region 113 is configured betweentwo insulating layers 112 a, 112 b in the third specific example. In thethird specific example, the heating elements 114 are characterized withresistances around 2Ω prior to annealing. After annealing at 400 F in aninert N₂ atmosphere, the resistances for the heating elements 114decreased 7% after 1 hour, with no substantial change in resistancefollowing additional annealing of 2 hours.

In a fourth specific example, the heating elements 114 comprise a 50 nmtitanium/tungsten adhesion material layer 116 and a 440 nm gold noblematerial layer 117 and were etched away in an identical pattern to thatin the first specific example, to form a surface including the heatingand sensing elements 114, 115. Similar to the first specific example,the heating elements 114 are defined by coarse patterning, and thesensing elements 115 are integrated with the coarse patterning of theheating elements 114 and defined by fine patterning. Also similar to thefirst specific example, the heating region 113 is configured between twoinsulating layers 112 a, 112 b in the fourth specific example. In thefourth specific example, the heating elements 114 are characterized withresistances around 40Ω and the sensing elements 115 are characterizedwith resistances around 100Ω prior to annealing. After annealing at 400F in an inert N₂ atmosphere, the resistances for both the heatingelements 114 and the sensing elements 115 decreased 11-14% after 1 hour,with no substantial further change in resistance following additionalannealing of 2 hours.

In the second, third, and fourth specific examples, analysis of theresistance values pre-annealing and post-annealing indicated that thecompositions and processing of the respective thin film layers 112, 116,117 significantly stabilize the resistance values and dynamics ofresistance behavior of the heating and sensing elements 114, 115 afterannealing. The third specific example with chromium and platinum layersdemonstrated substantially no variance from pre-anneal measurements ofthe electrical resistance after the second annealing step, and thefourth specific example with titanium, tungsten, and gold layersdemonstrated a 11%-14% reduction in resistance following the firstannealing step but then showed no additional variance after the secondannealing step. No further changes in resistance following stages ofannealing indicate that the dynamic behavior of the materials has beenarrested or completed (e.g., has reached equilibrium). The secondspecific example with chromium, platinum, and gold layers demonstrated aslightly higher variance (˜10%-30%) from pre-anneal resistance valuesindicating a higher level of dynamic behavior in these thin films, andfurther annealing was not completely sufficient to arrest the dynamicswith this combination. However, the combination of different adhesionand noble materials with the intermediate buffer layer(s) 119 to reduceor eliminate diffusion between adhesion layers 116 and noble layers 117,along with the annealing stage(s) produced positive effects with regardto stability of resistance behavior.

Other variations and examples of the heater-sensor dies 111 can compriseany suitable patterning configuration, any suitable arrangement ofinsulating layer(s) 112 and heating region(s) 113, any suitablecombination of adhesion, buffer, and/or noble layers 116, 119, 117,and/or any suitable annealing or other process that facilitatesstabilization of resistance behavior in the set of heater-sensor dies110.

1.1.1 Heater-Sensor Theory

In the specific examples described above, the microfabricatedheater-sensor dies 111 of the set of heater-sensor dies 110 areconfigured in a manner that produces rapid thermocycling for a givenlevel of power input. Design of the pattern 313 of heater-sensor dies111 in the specific examples was performed using a layout editor (e.g.,Mentor Graphics™ or L-Edit™), in order to generate the boustrophedonicstructures of the local morphology of the heater elements 114, withinthe bounds of the global circular morphology of the heater elements 114.Estimates of the electrical resistance of both thin-film heatingelements 114 and sensing elements 115 (e.g., RTDs) of the specificexamples was performed based upon a determination of sheet resistancesof the thin film layer(s) of the adhesion and noble layers 116, 117being deposited upon the insulating layer 112, with thicknesses of thethin film layers ranging from 50 nm-500 nm. In the specific example,sheet resistances were calculated based on the resistivity of the thinfilm and the width, thickness, and length of the thin film regions ofthe adhesion and noble layers 116, 117 being deposited. As such, in thespecific examples, the layouts of the heating and sensing element 114,115 patterns are configured to obtain desired heater-sensor coverage ona region (e.g., a region proximal to a surface of a sample container)being thermally cycled. In the specific examples, the configuration ofthe heater-sensor dies 111 also ensures that electrical resistancevalues of the heater-sensor dies are in within a desirable range, froman energy-use standpoint (e.g., a range that enables a range oftemperatures with a low input voltage).

Theoretically, the resistance(s) of the heating element(s) 114 arepreferably in the range of 50-100Ω to enable efficient heating of asubstrate with an input voltage from 20-30 volts; however, the heatingelements 114 can alternatively be characterized by other resistanceranges. A heating element 114 design characterized by a resistancebetween 50-100Ω and that uses an input voltage of 20-30 volts provides adissipation of 4-18 Watts of heat and is capable of rapidly heating adesired region of a thermally conductive substrate (e.g., siliconsubstrate). In addition, such a design parameters can produce atemperature across a 1 cm×1 cm substrate that equilibrates and producesa uniform temperature profile in a diffusion time of less than 1 second.Such a diffusion time thus enables well-controlled biochemicalreactions, with regard to temperature uniformity and control.

Theoretically, the resistance(s) of the sensing element(s) 115 (e.g.,RTD) are preferably in the range 200-300Ω, based upon correlating a“change in resistance” (dependent upon TCR) against the temperature ofthe sensing element 115. In one variation, the resistance behavior of anRTD can be characterized by the following equation:

R=R0*(1+α*ΔT+β*ΔT̂2)

where R=Resistance of an RTD at an unknown temperature (T);R0=Resistance of temperature at a reference temperature T0 (e.g., roomtemperature); α and β=experimentally determined constants; andΔT=Difference in temperature (T−T0). Under this equation, the change inresistance (ΔR) for an RTD per degree change (ΔT=1) is given byΔR=(α+β)*R0.

As such, for efficient temperature sensing as well as ensuring adetected temperature resolution of 0.1-0.2 C using RTDs, the resistancechange per degree change in temperature is preferably greater than˜0.1Ω. While higher changes in resistance can be desired, such higherchanges in temperature, for a given RTD material or thin filmcombination, results only when the initial resistance (R0) of the RTD issubstantially high. However, having a substantially high initialresistance increases the risk of the RTD self-heating during theresistance sensing process, thereby potentially causing additionalnoise/disturbances and inaccuracies in temperature measurement. The RTDsin a specific example of the system 100 were calibrated by measuring theresistance against 4-5 temperature points in the range the sensor isintended to be utilized. For biochemical assays, the typical range oftemperatures is room temperature (25° C.) to ˜100° C. By characterizingthe resistance (R) at each of several temperatures (T) within a range ofintended operation, one can obtain an experimental relationship for Rvs. T.

1.2 Other System Elements

As shown in FIGS. 1, 4A-4B, and 7A-7C, the system 100 can furthercomprise an electronics substrate 140 configured to couple heating andsensing elements of the set of heater-sensor dies to a controller 165, aset of heat-sink supports 150 configured to facilitate heat dissipationwithin the system 100, a set of elastic elements 160 configured to biasthe set of heater-sensor dies 110 against detection chambers for sampleprocessing, and can additionally comprise the controller 165 and/or acooling subsystem 170.

The electronics substrate 140 is preferably coupled to the set ofheater-sensor dies 110, and functions to enable communication betweenheating elements 114 and sensing elements 115 of each heater-sensor die111 in the set of heater-sensor dies no and a controller 165. As such,the electronics substrate preferably enables communication of heatoutput commands from the controller 165 to the heating element(s) 114,and communication of heating parameters (e.g., detected temperatures)sensed at the sensing element(s) 115 to the controller 165. Theelectronics substrate 140 preferably comprises a printed circuit board(PCB), and in one variation is a flexible PCB, as shown in FIG. 4A, inorder to facilitate contact between heater-sensor dies 111 in the set ofheater-sensor dies 110 and detection chambers (e.g., reaction vessels,sample containers, etc.) for processing of a sample according tomolecular diagnostic protocols. Alternatively, the PCB can be asubstantially rigid PCB or any other suitable PCB.

Preferably, the set of heater-sensor dies 110 is coupled to theelectronics substrate 140 in a manner that provides thermal and/orelectrical isolation of each heater-sensor dies 111 from the neighboringheater-sensor die(s) due to poor conductivity of the electronicssubstrate 140. However, the electronics substrate 140 and the set ofheater-sensor dies 110 can be configured in any alternative suitablemanner that provides isolation of each heater-sensor die 111. In somevariations, each heater-sensor die 111 is coupled to the electronicssubstrate by an adhesive layer 141 and/or a wire bond 142, as shown inFIG. 5A; however, each heater-sensor dies 111 can alternatively oradditionally be coupled to the electronics substrate 140 in any suitablemanner (e.g., using solder bonds in flip-chip bonding), as shown in FIG.5B. Furthermore, the system 100 can comprise any suitable number ofelectronics substrates.

The set of heat sink supports 150 is preferably coupled to at least oneof the set of heater-sensor dies 110 and the electronics substrate 140and functions to facilitate rapid thermocycling by dissipating heat fromthe set of heater-sensor dies 110 and/or the electronics substrate 140.The set of heat sink supports 150 can further function to providestructural support for the set of heater-sensor dies 110, such that theset of heater-sensor dies 110 is supported during compression (e.g.,compression against a set of detection chambers) and/or tension. In theabsence of heat sinking, the electronics substrate 140 and thesurrounding environment can potentially retain too much heat, whichcompromises the cooling of the set of heater-sensor dies 110. The set ofheat sink supports 150 can comprise multiple heat sink supports 151configured to define any suitable number of contact locations, or canalternatively comprise a single heat sink support 151 configured todefine any suitable number of contact locations. As shown in FIGS. 6Aand 6B, the system 100 preferably couples to a detection chamber (e.g.,reaction vessel, detection chamber) at a first side 101 a of thedetection chamber, as shown in FIG. 4A, which can restrict heatdissipation from the first side 101 a of the system. Furthermore, thesecond side 101 b of the detection chamber is typically used for opticalimaging for monitoring (e.g., realtime monitoring, delayed monitoring),and further limiting heat-sinking from the second side 101 b. Thus, itis preferable for the set of heat sink supports 150 to couple to thesystem 100 from a side of the system 100 that does not physicallyinterfere with optical imaging apparatus interfacing with the system100. However, alternative configurations of the set of heat sinksupports 150 can comprise coupling at any suitable side and/or anynumber of sides of the system 100.

As shown in FIGS. 6A and 6B, the set of heat sink supports 150 can beconfigured in any of a number of variations. In a first variation, eachheat sink support 151 can be directly placed against a first surface 105a of heater-sensor die 111 opposing that of a second surface 105 bcontacting a detection chamber, as shown in FIG. 6A. The first variationenables efficient transfer of heat out of the first surface 105 a of theheater-sensor die away from a respective detection chamber; however,excessive heat sinking can affect heating ramp rates. In a secondvariation, the system 100 comprises a thermally insulating assembly 152between a heater-sensor die 111 and a corresponding heat sink support151, as shown in FIG. 6B. In the second variation, the electronicssubstrate 140 can serve as the thermally insulating assembly 152 and canbe situated between the heater-sensor die 111 and a heat sink support151. Furthermore, in the second variation, a suitable thermal resistanceprovided by the electronics substrate 140 (e.g., through thickness,material selection, a combination of features) could produce a thermalcouple between the heater-sensor dies 111 and the heat sink support 151to permit the heating capacity of the heater-sensor die 111 to achievethe heating times and/or heating ramp rate required by the application,while still allowing adequate cooling rates. Additionally, the secondvariation can provide increased backside support to each of the set ofheater-sensor dies 110 as well as increased surface for adhesion.

In specific examples of the second variation, heat sinking andsupporting the “backside” of the electronics substrate 140 can beimplemented across multiple heater-sensor dies 111, separated by Societyfor Laboratory Automation and Screening (SLAS) standard spacings, suchas 9 mm, 4.5 mm or 2.25 mm spacings. The heat sink support 151 material(e.g., aluminum, copper, silver) in the specific examples is mated withthe electronics substrate 140 at each heater-sensor die location, withan air gap positioned laterally between each heater-sensor die location.This configuration can further function to reduce cross talk across aset of detection chambers in contact with the set of heater-sensor dies110. The set of heat sink supports 150 can, however, be configured inany other suitable manner to provide heat dissipation within the system100, without obstruction of optical detection apparatus, and withprovision of desired heat ramping and/or cycling behavior.

The set of elastic elements 160 is preferably coupled to a first surface104 a of the electronics substrate 140, as shown in FIG. 4A, andfunctions to promote contact between the set of heater-sensor dies 110and detection chambers (e.g., reaction vessels, detection chambers) forsample processing according to molecular diagnostic protocols. The setof elastic elements 160 can comprise any one more of springs andelastomeric elements, which can deform and provide transmit a biasingforce, through the electronics substrate 140, to reinforce contactbetween a set of detection chambers and the set of heater-sensor dies110. The set of elastic elements 160 can, however, additionally oralternatively include any other suitable elements configured to providea biasing force that reinforces contact between a set of detectionchambers and the set of heater-sensor dies 110 in an elastic or anon-elastic manner. In one such alternative variation, the system 100can include one or more actuators configured to drive each of the set ofheater-sensor dies 111 toward a corresponding detection chamber, and inanother such alternative variation, the system 100 can include a set ofmagnets (e.g., including magnet pairs surrounding the set ofheater-sensor dies 110 and a corresponding set of detection chambers),that function to reinforce coupling between the set of heater-sensordies 110 and the set of detection chambers. However, any other suitableelements can additionally or alternatively be used to facilitate uniformand consistent coupling between the set of heater-sensor dies 110 and aset of detection chambers.

In embodiments of the system 100 including a set of elastic elements160, the set of elastic elements 160 is preferably coupled to a firstsurface 104 a of the electronics substrate 140, such that each elasticelement in the set of elastic elements 160 facilitates contact between aheater-sensor dies 111 and a corresponding detection chamber. In a firstvariation, the set of elastic elements 160 is coupled to first surface104 a of a flexible PCB of the electronics substrate 140, as shown inFIG. 4A. In the first variation, contact between each heater-sensor dies111 and a corresponding detection chamber is thus maintained by abiasing force provided by an individual spring through the flexible PCBof the electronics substrate 140. In the first variation, the number ofelastic elements in the set of elastic elements 160 is equal to thenumber of heater-sensor dies in the set of heater-sensor dies 110, suchthat the set of elastic elements 160 and the set of heater-sensor dies110 are paired in a one-to-one manner. Alternatives to the firstvariation can, however, comprise any suitable number of elastic elementsin relation to a number of heater-sensor dies 110. In a secondvariation, the set of heater-sensor dies 110 is coupled to a secondsurface 104 b of a rigid PCB of the electronics substrate 140, with theset of elastic elements 160 coupled to the first surface 104 a of therigid PCB. In the second variation, the set of elastic elements 160 thusfunctions to collectively transfer a force through the rigid PCB tomaintain contact between the set of heater-sensor dies 110 and thedetection chambers. Alternatives to the second variation can alsocomprise any suitable number of springs in relation to a number ofheater-sensor dies in the set of heater-sensor dies 110. Furthermore,variations of the system 100 can include one or more elastic elementscoupled to any other elements directly or indirectly coupled to the setof heater-sensor dies 110. For instance, the system 100 can additionallyor alternatively include one or more springs 160′ coupled to basesurfaces of the set of heat-sink supports 150 interfacing with the setof heater-sensor dies, in order to transmit biasing forces.

As shown in FIGS. 1A and 1B, the system 100 can further comprise acontroller 165, which functions to automate and/or control heatingparameters provided by the set of heater-sensor dies 110. The controller165 can further be configured to provide heat parameter output commandsto the heating element(s) 114, and/or configured to receivecommunication of heating parameters (e.g., detected temperatures) sensedat the sensing element(s) 115 of the system 100. The controller 165preferably comprises a proportion-integral-derivative (PID) controller,but can alternatively be any other suitable controller 165. Thecontroller 165 preferably interfaces with the set of heater-sensor dies110 through the electronics substrate 140 by a connector; however, thecontroller 165 can interface with the set of heater-sensor dies 110 inany alternative suitable manner. Preferably, the controller 165 isconfigured to automate and control heat output parameters, including anyone or more of: heating temperatures, heating ramp rates, heating times(e.g., holding times), and any other suitable heating parameter(s).Furthermore, the controller 165 can be configured to control individualheater-sensor dies 111 in order to provide unique heating parameters forindividual detection chambers and/or can be configured to provide commonheating parameters for all heater-sensor dies 111 in the set ofheater-sensor dies 110. In a specific example, the controller 165comprises a Yokogawa UT750 PID controller, an Arduino UNO R3microcontroller configured to cycle the UT750 through temperature stagesand to control temperature holding, a resistance-to-voltage conversioncircuit, and two power supplies—a first power supply configured tosupply power to the set of heater-sensor dies 110 and a second powersupply configured to supply voltage to the resistance-to-voltageconversion circuit. In the specific example, the controller 165comprises a resistance-to-voltage conversion circuit because the UT750PID controller requires voltage as an input for PID control. In anotherspecific example, the controller 165 comprises a National InstrumentsLabView based system comprised of an NI cDAQ-9178 chassis with an NI9219 universal analog input card and an NI 9485 eight-channelsolid-state relay sourcing or sinking digital output module solid-staterelay card. In this specific example, the cDAQ-9178 supports the NI 9219and NI 9485 cards, the NI 9219 is used to obtain the RTD inputs, and theNI 9485 cycles the power supply voltage to individual heater-sensor diesof the set of heater-sensor dies 110. Further, in this specific example,the controller 165 is expandable to 12 or more channels through the useof additional NI 9219 and NI 9485 cards, each of which can handleseveral channels.

As shown in FIGS. 8A and 8B, the system 100 can further comprise acooling subsystem 170, which functions to provide heat transfer from thesystem 100 in order to further enhance controlled heating and cooling bythe system 100. The cooling subsystem 170 is preferably configured toprovide at least one of convective cooling and conductive cooling of thesystem 100, but can alternatively be configured to provide any othersuitable cooling mechanism or combination of cooling mechanisms. In onevariation, the cooling subsystem 170 can comprise a fan 171 thatprovides convective heat transfer from the system 100. In thisvariation, the fan 171 can be coupled to any suitable element of thesystem 100, such as the set of heat sink supports 150, as shown in A.Furthermore, alternatives to this variation can comprise any suitablenumber of fans of any suitable dimension and configuration, examples ofwhich are shown in FIGS. 7A and 7B. In one such example, the system caninclude a set of cooling elements integrated with each heat sink supportof the set of heat sink supports. In another variation, the coolingsubsystem 170 can additionally or alternatively comprise a Peltierdevice, as shown in FIG. 7C. The Peltier device can be cooled andmaintained at a defined temperature (e.g., in the 10-25 C range) toprovide a substantial temperature gradient for cooling during a thermalcycling process, which can decrease cooling times and/or cycle times. Inyet another variation, the cooling subsystem 170 can additionally oralternatively comprise a liquid cooling system (e.g., water coolingsystem) configured to surround and absorb heat from one or moreheater-sensor dies of the set of heater-sensor dies 110, for instance,by way of the set of heat sink supports 150. The cooling subsystem 170can additionally or alternatively comprise any other suitable coolingelement(s).

In some variations, reflection from the set of heater-sensor dies 110can interfere with light transmitted to photodetectors of an opticalsubsystem opposed (e.g., directly opposed, in opposition) to the set ofheater-sensor dies 110 (e.g., light emitted from the set of biologicalsamples, light transmitted through filters of an optical subsystem),especially in configurations wherein a set of detection chambers isconfigured between the set of heater-sensor dies and optical elements ofan optical subsystem. In these variations, the set of heater-sensor dies110 can include elements that reduce or eliminate reflection from anyportion of the set of heater-sensor dies (e.g., reflection from theheating region, etc.), thereby facilitating analysis of a set ofbiological samples within the set of detection chambers. In onevariation, the set of heater-sensor dies 110 can include or be coupledto one or more non-reflective coatings 180 at surfaces of the set ofheater-sensor dies 110 upon which light from the optical subsystemimpinges. In a specific example, the non-reflective coating 180 cancomprise a high-temperature paint (e.g., dark paint, flat paint) thatfunctions to absorb and/or diffuse light from an opposing opticalsubsystem, while facilitating heat transfer to a set of detectionchambers in thermal communication with the set of heater-sensor dies110. In another variation, the set of heater-sensor dies 110 can beconfigured to be in misalignment with photodetectors of the opticalsubsystem, such that reflection does not interfere with lighttransmitted to the photodetectors of the optical subsystem. In oneexample, the set of heater-sensor dies 110 can be configured to heat aset of detection chambers from a first direction, and the opticalsubsystem can be configured to receive light from the set of detectionchambers from a second direction (e.g., a direction non-parallel to thefirst direction), such that reflection from the set of heater-sensordies 110 does not cause interference. In still other variations, the setof heater-sensor dies 110 can include any other suitable elements (e.g.,coatings, layers, etc.) and/or be configured in any other suitablemanner that eliminates, prevents, or mitigates reflection from the setof heater-sensor dies 110 from interfering with light transmitted tophotodetectors of an optical subsystem in opposition to the set ofheater-sensor dies 110.

Variations of the system 100 can, however, include any other suitableelement(s) configured to provide uniform, accurate, precise, andreliable heating of one or more detection chambers in thermalcommunication with the system 100. Furthermore, as a person skilled inthe art will recognize from the previous detailed description and fromthe figures, modifications and changes can be made to the preferredembodiments of the system 100 without departing from the scope of thesystem 100.

2. Method of Manufacture

As shown in FIG. 8, a method 200 of manufacturing a system forthermocycling biological sample within detection chambers comprises:forming a first insulating layer coupled to exposed surfaces of asubstrate S210, depositing an adhesion material layer of a heatingregion onto the first insulating layer of the substrate S220, depositinga noble material layer of the heating region onto the adhesion materiallayer S230; removing material of the heating region S240, therebyforming a pattern of voids that defines a coarse pattern associated witha heating element of the heating region and a fine pattern, integratedinto the coarse pattern and associated with a sensing element of theheating region; forming a second insulating layer coupled to the heatingregion and to the first insulating layer by way of the pattern of voidsS250; and annealing the first insulating layer, the heating region, andthe second insulating layer for a first duration S260.

The method 200 functions to generate heating and sensing elements of athermocycling system that can provide rapid thermocycling withoutsignificant power requirements, while ensuring a suitably closecorrelation between an actual heating temperature and a temperatureset-point (i.e., an intended heating temperature). In some embodiments,the method 200 can function to produce a heating and sensing element ofan integrated heater-sensor die as described in Section 1 above, whichcan controllably and individually heat small sample volumes (e.g.,picoliters, nanoliters, microliters). Furthermore, the method 200 ofmanufacture preferably implements a priori predictions of electricalresistance values of thin film combinations of the set of heater-sensordies 110, as described in Section 1 above, and accounts for and/orprevents signal drift in relation to actual and intended heatingtemperatures, to maintain controlled sample heating.

Block S210 recites: forming a first insulating layer coupled to exposedsurfaces of a substrate, which functions to electrically isolateportions of a heating region from other elements of the thermocyclingsystem. The substrate is preferably a silicon substrate, but canalternatively be any other suitable semi-conducting, or non-conductingsubstrate. As such, in variations, the substrate can be composed of asemi-conducting material (e.g., silicon, quartz, gallium arsenide),and/or an insulating material (e.g., glass, ceramic). In somevariations, the substrate 130 can even comprise a combination ofmaterials (e.g., as in a composite, as in an alloy). In examples whereinthe substrate is a silicon substrate, the substrate can be composed ofsilicon with any suitable type (e.g., P-type), doping (e.g.,boron-doping), miller index orientation, resistivity, thickness, totalthickness variation, and/or polish.

In forming the first insulating layer, Block S210 can be performed usingany one or more of: thermal oxide growth, chemical vapor deposition(CVD), spin coating, spray coating, and any other suitable method ofdepositing a localized layer of an insulating material. Preferably, thefirst insulating layer is composed of an insulating oxide material, andin examples can include any one or more of: a thermally grown siliconoxide, a chemical vapor deposited oxide, a deposited titanium oxide, adeposited tantalum oxide, and any other suitable oxide grown and/ordeposited in any other suitable manner. However, the first insulatinglayer can additionally or alternatively include an insulating polymer(e.g., a polyimide, a cyanate ester, a bismaleimide, a benzoxazine, aphthalonitrile, a phenolic, etc.) that is chemical and heat resistantand/or any other suitable material (e.g., chemical vapor depositednitride, other nitride, paralene, etc.) that is configured to providethe first insulating layer.

In one example of Block S210, the first insulating layer comprises anoxide material, and is formed by growing the oxide material on asubstrate. In one example of Block S210, the insulating layer comprisesa 0.2 mm layer of silicon oxide, and is formed on a 100 mm silicon waferusing thermal oxidation at 900° C. using water vapor (i.e., in wetoxidation) or oxygen (i.e., in dry oxidation) as the oxidant. Inalternative variations and examples of Block S210, the first insulatinglayer can be formed using high or low temperature thermal oxidation,using any suitable oxidant, and/or using any other suitable method(e.g., fluid deposition of an electrically insulating polymer,softbaking/hardbaking of a deposited polymer, etc.).

Block S220 recites: depositing an adhesion material layer of a heatingregion onto the first insulating layer of the substrate, which functionsto facilitate bonding of a noble material layer of the heating region tothe first insulating layer as in Block S230. The adhesion materialpreferably comprises an adhesion material that is characterized asactive in reacting with the first insulating layer, in order tofacilitate coupling between the first insulating layer and the adhesionmaterial. However, the adhesion material of the adhesion material layercan additionally or alternatively have any other suitablecharacteristic(s). In variations wherein the first insulating layercomprises an insulating oxide, the adhesion materials used in theadhesion material layer(s) can comprise materials that are oxygen-activeto chemically react with an oxide surface (e.g., materials that have ahigh heat of oxide formation). In examples of oxygen-active materials,the adhesion material layer can be composed of any one or more of:chromium, titanium, niobium, vanadium, any other suitable adhesionmaterial that reacts with the insulating layer, and any combination oralloy of any of the above materials.

In Block S220, the adhesion material layer is preferably coupled to thefirst insulating layer by one or more of evaporation and sputtering.However, in alternative variations, the adhesion material layer can becoupled to the first insulating layer by any one or more of: deposition(e.g., electrodeposition, CVD, etc.), plating (e.g., chemical plating,electroplating), and any other suitable process of coupling the adhesionmaterial layer to the first insulating layer. Furthermore, in exampleswherein the adhesion material layer is evaporated or sputtered, theinsulating layer-substrate subassembly generated in Block S210 can betranslated or rotated in order to facilitate uniform deposition of theadhesion material layer. In Block S220, the adhesion material layer ispreferably processed to a thickness of under 100 nm; however, theadhesion material layer can alternatively be processed to any othersuitable thickness. In specific examples, the adhesion material layercomprises a 50 nm thick layer of chromium, or a 50 nm thick layer of acombination of titanium and tungsten.

Block S230 recites: depositing a noble material layer of the heatingregion onto the adhesion material layer, which functions to form aportion of a heating region comprising a heating element and a sensingelement of the system. The noble material preferably comprises a noblematerial that is characterized as having good thermal conductivity andaffinity to coupling with the adhesion material layer in a reliablemanner during thermocycling of the system. The noble material layer canalternatively have characteristics including any one or more of:characteristics that inhibit diffusion between the adhesion materiallayer and the noble material layer, high fatigue resistance, highfracture resistance, and any other suitable property that providesreliability during thermocycling of the system. In examples of noblematerials that operate well with examples of adhesion materialsdescribed above, the noble material layer can be composed of any one ormore of: gold, platinum, tungsten, palladium, any other noble materialthat interacts well with the adhesion material layer, and anycombination or alloy of any of the above materials.

In Block S230, the noble material layer is preferably coupled to theadhesion material layer by one or more of evaporation and sputtering.However, in alternative variations, the noble material layer can becoupled to the adhesion material layer by any one or more of: deposition(e.g., electrodeposition, CVD, etc.), plating (e.g., chemical plating,electroplating), and any other suitable process of coupling the noblematerial layer to the adhesion material layer. Furthermore, in exampleswherein the noble material layer is evaporated or sputtered, theadhesion material layer—insulating layer-substrate subassembly generatedin Block S220 can be translated or rotated in order to facilitateuniform deposition of the noble material layer. In Block S230, the noblematerial layer is preferably processed to a thickness of under 500 nm;however, the noble material layer can alternatively be processed to anyother suitable thickness. In specific examples, the noble material layercomprises a 200 nm thick layer of gold, a 300 nm thick layer of gold, a440 nm thick layer of gold, and/or a 100 nm thick layer of platinum.

In some variations, the method 200 can further include Block S225, whichrecites: depositing an intermediate buffer layer configured between theadhesion material layer and the noble material layer. Block S225functions to provide a layer that reduces or prevents diffusion betweenthe noble material layer and the adhesion material layer, thus improvingdynamic stability (e.g., in electrical resistance behavior) of thesystem. In variations, the intermediate buffer layer thus hascharacteristics that contribute to behavior as a barrier againstdiffusion between the adhesion material layer and the noble materiallayer. In examples, the intermediate buffer layer comprises platinumand/or titanium, and can be processed onto an adhesion material layer byone or more of evaporation and sputtering. However, in alternativevariations, the intermediate buffer layer can be coupled to the adhesionmaterial layer by any one or more of: deposition (e.g.,electrodeposition, CVD, etc.), plating (e.g., chemical plating,electroplating), and any other suitable process of coupling theintermediate buffer layer to adhesion material layer, prior to couplingof noble material layer to the intermediate buffer layer. Theintermediate buffer layer is preferably processed to a thickness of lessthan 150 nm, and in specific examples can comprise a 100 nm thick layerof platinum, or a 50 nm thick layer of titanium. However, theintermediate buffer layer can alternatively have any other suitablethickness. Furthermore, variations of the heating region 113 comprisingadhesion material layers and noble material layers can include anysuitable number of adhesion material layers, noble material layers, andintermediate buffer layers that prevent diffusion between an adhesionmaterial layer into an adjacent noble material layer, wherein each layercan have any other suitable thickness.

Block S240 recites: removing material of the heating region, whichfunctions to form a pattern of voids that defines a coarse patternassociated with a heating element of the heating region and a fineregion, integrated into the coarse pattern and associated with a sensingelement of the heating region. Block S240 functions to produce elementsof the heating region with desired resistance behavior andcharacteristics, in order to provide reliable and accurate heatparameter sensing and output within the system. Block S240 preferablyprovides one or more heating elements and sensing elements integratedwith the heating elements, wherein the heating element(s) and thesensing element(s) have desired theoretical resistance characteristicsas described in Section 1.1.1 above; however, Block S240 canalternatively provide heating elements and sensing elements with anyother suitable resistance characteristics.

Block S240 preferably produces a pattern of voids having geometricfeatures (e.g., width, thickness, length, spacing) that facilitateuniform heating and provide desired heating and sensing characteristics(e.g., resistance characteristics). In some variations, the pattern ofvoids can define any one or more of: linear segments, non-linearsegments, boustrophedonic segments, continuous segments, non-continuoussegments, and any other suitable segment(s) having any other suitablegeometry (i.e., width, depth, height, length, path, etc.) within anassembly of the adhesion material layer(s), noble material layer(s),and/or intermediate buffer layer(s) produced in Blocks S220, S230 and/orS225. The pattern of voids produced in Block S240 can further besymmetric about any suitable reference (e.g., reference line, referenceplane, etc.), or can alternatively be non-symmetric. Furthermore, insome variations, the pattern of voids can define a global morphology(e.g., circular footprint, ellipsoidal footprint, polygonal footprint,etc.) at a first scale (e.g., macroscopic scale) but have a localmorphology at a second scale (e.g., microscopic scale), wherein thelocal morphology provides desired characteristics (e.g., resistancecharacteristics) that are attributed to elements (e.g., heatingelements, sensing elements) of the heating region. As such, the globalmorphology can provide conformation (e.g., in shape) between the heatingregion and a detection chamber configured to contact the heating region,and the local morphology can provide uniform heating and/or accuratesensing of heating parameters by utilizing structural features having asmaller governing dimension.

The pattern of voids is preferably defined entirely through the assemblyof the adhesion material layer(s), noble material layer(s), and/orintermediate buffer layer(s) produced in Blocks S220, S230 and/or S225(e.g., through the assembly to the first insulating layer); however, thepattern of voids can alternatively be defined to any other suitablethickness through the assembly. Preferably, Block S240 includesproducing the pattern of voids by way of a lithographic process (e.g.,lithographic process with a positive resist, lithographic process with anegative resist). However, producing the pattern of voids in Block S240can additionally or alternatively be performed using any one or more of:etching, punching, die-cutting, water cutting, laser cutting, and anyother suitable method of removing material. In one example, the assemblycomprising the adhesion material layer and the noble material layerproduced in examples of Blocks S220 and S230 can be covered withpositive photoresist (e.g., a photomask designed according to theheating pattern) and lithographically etched in exposed regions. In theexample, the positive photoresist can then be removed to reveal thepattern of voids. In other variations of the example, the pattern ofvoids can be produced using any lithographic method, using positiveand/or negative etching to form the heating pattern, and/or using anyother suitable method. In one example of an alternative implementationof Step S240′, the pattern of voids can be produced using a lift-offprocess, wherein a sacrificial layer is used to define the pattern ofvoids, the heating region material(s) is (are) deposited, and then thesacrificial layer is removed to reveal the heating pattern.

In a specific example of Block S240, an assembly comprising an adhesionmaterial layer, a noble material layer, and an intermediate buffer layerconfigured between the adhesion material layer and the noble materiallayer is configured to have a global footprint defining circular regionhaving a diameter of 5 mm. The pattern of voids, produced by alithographic process, defines three heating elements including: acentral circular heating element and two circumferential heatingelements, as shown in FIG. 3, configured to form a boundary with aserpentine-shaped pattern about the central circular heating element. Inthe specific example, the two circumferential heating elements are eachconfigured to form a semicircular perimeter about the central circularheating element. The pattern of voids produced in the specific exampleof Block S240 further comprises three integrated sensing elements (i.e.,resistance temperature sensors, RTDs) distributed at three locationswithin the 5 mm circular footprint. In the specific example, the patternof voids was designed using a layout editor (e.g., Mentor Graphics™ orL-Edit™), to define the heating elements and the sensing elementsaccording to desired resistance characteristics as described in Section1.1.1 above. The specific example of Block S240 produced heatingelements having coarse patterning, and sensing elements 115 integratedwith the heating elements and having fine patterning, as shown in FIGS.3A and 3B. Variations of the specific example of Block S240 can,however, generate any other suitable pattern of voids to define theheating element(s) and the sensing element(s) in any other suitablemanner.

Blocks S210, S220, S230, and S240 are preferably performed in the orderdescribed; however, Blocks S210, S220, S230, and S240 can alternativelybe performed in any other suitable order. For instance, in one variationthe patterned assembly comprising the adhesion material layer(s), thenoble material layer(s), and/or the intermediate buffer layer(s) can beformed by any suitable method (e.g., molding, laser cutting, stamping,3D printing, stereolithography, etc.) and then coupled to the firstinsulating layer according to any suitable manner.

Block S250 recites: forming a second insulating layer coupled to theheating region and to the first insulating layer by way of the patternof voids, which functions to electrically isolate portions of a heatingregion from other elements of the thermocycling system, and to furtherprovide stability within the system. In forming the second insulatinglayer, Block S250 is preferably performed using a deposition process(e.g., CVD); however, Block S250 can additionally or alternatively beperformed using any one or more of: thermal oxide growth, spin coating,spray coating, and any other suitable method of depositing a localizedlayer of an insulating material. Preferably, the second insulating layeris composed of an insulating oxide material, and in examples can includeany one or more of: a silicon oxide (e.g., deposited by CVD, thermallygrown), a titanium oxide, a tantalum oxide, and any other suitable oxidegrown and/or deposited in any other suitable manner. However, the firstsecond layer can additionally or alternatively include an insulatingpolymer (e.g., a polyimide, a cyanate ester, a bismaleimide, abenzoxazine, a phthalonitrile, a phenolic, etc.) that is chemical andheat resistant and can be processed onto the first insulating layer andthe heating region by any one or more of: casting (e.g., drop casting),printing, dipping, and using any other suitable method. The secondinsulating layer processed in Block S250 can additionally oralternatively comprise any other suitable material (e.g., chemical vapordeposited nitride, other nitride, paralene, etc.) that is configured toprovide the second insulating layer.

Block S260 recites: annealing the first insulating layer, the heatingregion, and the second insulating layer for a first duration, whichfunctions to stabilize dynamic resistance behavior of heating andsensing elements of the system. Since a majority of changes inresistance (e.g., drift) come from thermal reactions, settling,diffusion, and improved adhesion, annealing can accelerate the processesthat lead to changes in resistance, thus producing a system with stableresistance behavior (e.g., stable for 3-5 years) by driving dynamicprocesses toward an equilibrium state (or other stable state) prior touse of the system in sample-processing applications. In Block S260,annealing can comprise a single stage of annealing or multiple stages ofannealing, and can additionally or alternatively comprise differentconditions (e.g., temperatures, durations, environmental conditions)during the annealing stage(s). Preferably, annealing thus producesstable resistances that are substantially low (in order to enabledriving of the system at low voltages) and have limited variability,indicating that underlying material dynamics have reached equilibrium.

In Block S260, annealing is preferably performed at constanttemperature, and in variations, can be performed at a temperaturebetween 300 C and 600 C (or any other suitable temperature below amelting temperature of the noble material layer and the adhesionmaterial layer); however, annealing can alternatively be performed atany other suitable temperature or range of temperatures, and canadditionally or alternatively be performed at a non-constant temperature(e.g., cyclically varying temperature, non-cyclically varyingtemperature). Annealing is preferably performed in an inert atmosphere(e.g., inert N₂ atmosphere) to control reactions between the atmosphereand elements being annealed; however, annealing can alternatively beperformed in any other suitable atmosphere. In a specific example, BlockS240 includes annealing the first insulating layer, the heating region,and the second insulating layer at 400 F in an inert N₂ atmosphere forone hour, which can, in some variations of the specific example, beextended for up to 2 hours following the first hour of annealing.

As shown in FIG. 8, the method 200 can further include Block S270, whichrecites: coupling a non-reflective coating to at least one heater-sensordie of the set of heater-sensor dies. Block S270 functions to process atleast a subset of the set of heater-sensor dies 110 so that they do notinterfere with light transmitted to photodetectors of an opticalsubsystem opposed (e.g., directly opposed, in opposition) to the set ofheater-sensor dies 110 (e.g., light emitted from the set of biologicalsamples, light transmitted through filters of an optical subsystem),especially in configurations wherein a set of detection chambers isconfigured between the set of heater-sensor dies and optical elements ofan optical subsystem. The non-reflective coating is preferably coupledidentically to all heater-sensor dies of the set of heater sensor dies;however, the non-reflective coating can alternatively be couplednon-identically to one or more heater-sensor dies of the set ofheater-sensor dies. As such, in variations, one or more subsets of theset of heater-sensor dies can be coupled to non-reflective coatings in amanner that provides different light reflection properties for thesubset(s) of the set of heater-sensor dies.

In Block S270, the non-reflective coating is preferably a material layerthat is applied superficial to at least one of the first insulatinglayer and the second insulating layer processed in variations of BlocksS210 and S250, respectively. In one example, the non-reflective coatingprocessed in Block S270 can comprise a high-temperature paint (e.g.,dark paint, flat paint) that functions to absorb and/or diffuse lightfrom an opposing optical subsystem, while facilitating heat transfer toa set of detection chambers in thermal communication with the set ofheater-sensor dies. In this example, the high-temperature paint can beapplied by any one or more of: brushing, spraying, dipping, printing,and any other suitable method of coupling the high-temperature paint toone or more surfaces of at least a subset of the set of heater-sensordies. However, the non-reflective coating can alternatively be processedsimultaneously with or can comprise one or more of the first insulatinglayer and the second insulating layer processed in variations of BlocksS210 and S260, respectively. For instance, one or more of the first andthe second insulating layer can include a modified oxide layer that haslow-reflectivity, thus preventing interference caused by light reflectedfrom the set of heater-sensor dies. In some extreme variations, however,mitigation of interference due to reflected light from the set ofheater-sensor dies can be produced by configuring the set ofheater-sensor dies to be in misalignment with photodetectors of theoptical subsystem, such that reflection does not interfere with lighttransmitted to the photodetectors of the optical subsystem, in modifiedversions of Block S270. For instance, the set of heater-sensor dies canbe configured to heat a set of detection chambers from a firstdirection, and the optical subsystem can be configured to receive lightfrom the set of detection chambers from a second direction (e.g., adirection non-parallel to the first direction), such that reflectionfrom the set of heater-sensor dies 110 does not cause interference. Instill other variations of Block S270, the set of heater-sensor dies canbe processed with any other suitable elements (e.g., coatings, layers,etc.) and/or be configured in any other suitable manner that eliminates,prevents, or mitigates reflection from the set of heater-sensor diesfrom interfering with light transmitted to photodetectors of an opticalsubsystem in opposition to the set of heater-sensor dies 110.

The method 200 can additionally or alternatively include any othersuitable blocks or steps configured to facilitate fabrication of athermocycling element that can heat small volumes (e.g., microlitervolumes, nanoliter volumes, picoliter volumes) of biological sampleswithin containers in contact with the thermocycling element. Forinstance, the method 200 can include any one or more of: coupling theheating region to an electronics substrate S280; coupling theelectronics substrate to an elastic element configured to bias thesystem against a detection chamber S290; and coupling at least one ofthe electronics substrate and the heating region to a heat sink-supportS300 that facilitates heat dissipation within the system, whileproviding mechanical support for the heating region within the system.The method 200 can, however, include any other suitable blocks or steps.

The system 100 and/or method 200 of the preferred embodiment andvariations thereof can be embodied and/or implemented at least in partas a machine configured to receive a computer-readable medium storingcomputer-readable instructions. The instructions are preferably executedby computer-executable components preferably integrated with the system300 and one or more portions of the processor 350. The computer-readablemedium can be stored on any suitable computer-readable media such asRAMs, ROMs, flash memory, EEPROMs, optical devices (CD or DVD), harddrives, floppy drives, or any suitable device. The computer-executablecomponent is preferably a general or application specific processor, butany suitable dedicated hardware or hardware/firmware combination devicecan alternatively or additionally execute the instructions.

The FIGURES illustrate the architecture, functionality and operation ofpossible implementations of methods according to preferred embodiments,example configurations, and variations thereof. In this regard, eachblock in the flowchart or block diagrams may represent a module,segment, or portion of code, which comprises one or more executableinstructions for implementing the specified logical function(s). Itshould also be noted that, in some alternative implementations, thefunctions noted in the block can occur out of the order noted in theFIGURES. For example, two blocks shown in succession may, in fact, beexecuted substantially concurrently, or the blocks may sometimes beexecuted in the reverse order, depending upon the functionalityinvolved. It will also be noted that each block of the block diagramsand/or flowchart illustration, and combinations of blocks in the blockdiagrams and/or flowchart illustration, can be implemented by specialpurpose hardware-based systems that perform the specified functions oracts, or combinations of special purpose hardware and computerinstructions.

As a person skilled in the art will recognize from the previous detaileddescription and from the figures and claims, modifications and changescan be made to the preferred embodiments of the invention withoutdeparting from the scope of this invention defined in the followingclaims.

We claim:
 1. A system for thermocycling biological samples detectionchambers comprising: a set of heater-sensor dies, each heater-sensor diein the set of heater-sensor dies comprising: an assembly including afirst insulating layer, a heating region comprising an adhesion materiallayer coupled to the first insulating layer and a noble material layercoupled to the adhesion material layer, and a second insulating layercoupled to the heating region and to the first insulating layer througha pattern of voids in the heating region, wherein the pattern of voidsin heating region defines a coarse pattern associated with a heatingelement of the heating region and a fine pattern, integrated into thecoarse pattern and associated with a sensing element of the heatingregion; an electronics substrate configured to couple heating elementsand sensing elements of the set of heater-sensor dies to a controller;and a set of elastic elements coupled to a second substrate surface ofthe electronics substrate opposing a first substrate surface of theelectronics substrate interfacing with the assemblies of the set ofheater-sensor dies and configured to bias each of the set ofheater-sensor dies against a detection chamber in a configurationwherein the set of heater-sensor dies is in thermal communication with aset of detection chambers.
 2. The system of claim 1, wherein theadhesion material layer comprises at least one of chromium, titanium,niobium, and vanadium, and the noble material layer comprises at leastone of gold, platinum, and tungsten, palladium.
 3. The system of claim1, wherein the adhesion material layer and the noble material layer areannealed layers configured to provide stabilized resistance behavior andprevent drift in heating and sensing using the heating region.
 4. Thesystem of claim 2, wherein the assembly further comprises anintermediate buffer layer composed of at least one of platinum andtitanium, situated between the adhesion material layer and the noblematerial layer, and configured to provide a barrier against diffusionbetween the adhesion material layer and the noble material layer.
 5. Thesystem of claim 2, wherein the adhesion material layer has a thicknessless than or equal to 100 nm, and the noble material layer has athickness less than or equal to 500 nm.
 6. The system of claim 2,wherein the pattern of voids defines a pattern of boustrophedonicsegments including wide segments of the coarse pattern associated withthe heating element and segments of the fine pattern, surrounded by andnarrower than the wide segments, associated with the sensing element ofthe heating region.
 7. The system of claim 6, wherein the patterndefined through the adhesion material layer and the noble material layeris configured to provide a resistance change per temperature changegreater than 0.1 ohms/° C.
 8. The system of claim 7, wherein theelectronics substrate is a flexible substrate and wherein the systemfurther comprises a set of heat-sink supports coupled to at least one ofthe set of heater-sensor dies and the second substrate surface of theelectronics substrate and configured to dissipate heat generated by theset of heater-sensor dies, wherein a base surface of each of the set ofheat-sink supports is coupled to an elastic element configured totransmit a biasing force through the flexible substrate, therebymaintaining thermal communication between the set of heater-sensor diesand the set of detection chambers in the configuration wherein the setof heater-sensor dies is in thermal communication with the set ofdetection chambers.
 9. The system of claim 1, wherein each of the set ofheater-sensor dies includes a coating, proximal the heating surface,configured to mitigate reflection of light from the heating surfacetoward photodetectors of an optical subsystem, in a configurationwherein the set of heater-sensor dies is opposed to photodetectors ofthe optical subsystem.
 10. A method of manufacturing a system forthermocycling biological samples within detection chambers, the methodcomprising: at a substrate, forming a first insulating layer coupled toexposed surfaces of the substrate; depositing an adhesion material layerof a heating region onto the first insulating layer of the substrate;depositing a noble material layer of the heating region onto theadhesion material layer; removing material of the heating region,entirely to the first insulating layer, thereby forming a pattern ofvoids that defines a coarse pattern associated with a heating element ofthe heating region and a fine pattern, integrated into the coarsepattern and associated with a sensing element of the heating region;forming a second insulating layer coupled to the heating region and tothe first insulating layer by way of the pattern of voids; and annealingthe first insulating layer, the heating region, and the secondinsulating layer for a first duration.
 11. The method of claim 10,wherein depositing the adhesion material layer comprises depositing anoxygen-active material, comprising at least one of chromium, titanium,niobium, and vanadium, upon the first insulating layer.
 12. The methodof claim 11, wherein depositing the adhesion material layer comprises atleast one of sputtering and evaporating the oxygen-active material to athickness of less than or equal to 100 nm.
 13. The method of claim 11,wherein depositing the noble material layer comprises depositing atleast one of gold, platinum, and tungsten, palladium superficial to theadhesion material layer.
 14. The method of claim 10, further comprisingdepositing an intermediate buffer layer, comprising at least one ofplatinum and titanium, configured between the adhesion material layerand the noble material layer, wherein the intermediate buffer layerprovides a barrier against diffusion between the adhesion material layerand the noble material layer.
 15. The method of claim 10, whereinforming the pattern of voids comprises lithographically etchingboustrophedonic segments across the adhesion material layer and thenoble material layer, including wide segments of the coarse patternassociated with the heating element and segments of the fine pattern,surrounded by and narrower than the wide segments, associated with thesensing element of the heating region.
 16. The method of claim 15,wherein forming the pattern of voids comprises defining a centralcircular heating element, a circumferential heating element with aserpentine-shaped path at least partially surrounding the centralcircular heating element, and a sensing element integrated within thecentral circular heating element.
 17. The method of claim 10, whereinforming the first insulating layer comprises inducing thermal oxidegrowth at the substrate, and wherein forming the second insulating layercomprises depositing an oxide material upon the first heating region andthe first insulating layer.
 18. The method of claim 10, whereinannealing the first insulating layer, the heating region, and the secondinsulating layer comprises annealing at a temperature from 300° C.-600°C. within an inert N₂ atmosphere.
 19. The method of claim 18, whereinannealing comprises annealing the first insulating layer, the heatingregion, and the second insulating layer and monitoring resistance of theheating region until material dynamics of the heating region reachequilibrium.
 20. The method of claim 10, further comprising coupling acoating, proximal to the heating surface of at least one heater-sensordie of the set of heater-sensor dies, wherein the coating is configuredto mitigate reflection of light from the heating surface towardphotodetectors of an optical subsystem, in a configuration wherein theset of heater-sensor dies is opposed to photodetectors of the opticalsubsystem.